Studies are determining the location and possible metabolic role of oligo(U) sequences in nuclear RNA (hnRNA) and mRNA metabolism and the mechanism by which DRB(5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) causes premature transcription termination of mouse beta-Hb mRNA. Procedures were developed which allowed, for the first time, the separation of HeLa cell oligo(U+) hnRNA from oligo(U-) hnRNA. It was also shown that the fraction of hnRNA (approximately 30%) which was resistant to DRB inhibition had the same oligo(U) content as control hnRNA. Murine erythroleukemic cell (MELC) hnRNA was shown to contain oligo(U) sequences identical in size to the oligo(U) in HeLa hnRNA. MELC oligo(U+) hnRNA was separated from oligo(U-) hnRNA and characterized. The preparation of polyadenylated Sepharose, which has extremely low nonspecific binding and was stable for many months, was described. DRB was shown to inhibit the formation of cytoplasmic beta-Hb mRNA in differentiating MELC by approximately 90%. This inhibition was the result of premature transcription termination within the promoter proximal region of the beta-Hb gene; this is the first report of premature termination occurring in a cellular gene. Measurements of the accumulation of [3H]DRB as well as studies employing wild-type mouse lymphoma cells and mutants deficient in nucleoside transport indicated that the inhibition of mRNA transcription by DRB did not involve the accumulation or metabolism of DRB. This suggested that inhibition of mRNA synthesis by DRB may result from secondary event(s) triggered by interaction of DRB with a surface membrane component or may require the transport and accumulation of only minute amounts of DRB.